17-Oxime ethers of oxidized ecdysteroid derivatives modulate oxidative stress in human brain endothelial cells and dose-dependently might protect or damage the blood-brain barrier

20-Hydroxyecdysone and several of its oxidized derivatives exert cytoprotective effect in mammals including humans. Inspired by this bioactivity of ecdysteroids, in the current study it was our aim to prepare a set of sidechain-modified derivatives and to evaluate their potential to protect the blood-brain barrier (BBB) from oxidative stress. Six novel ecdysteroids, including an oxime and five oxime ethers, were obtained through regioselective synthesis from a sidechain-cleaved calonysterone derivative 2 and fully characterized by comprehensive NMR techniques revealing their complete 1H and 13C signal assignments. Surprisingly, several compounds sensitized hCMEC/D3 brain microvascular endothelial cells to tert-butyl hydroperoxide (tBHP)-induced oxidative damage as recorded by impedance measurements. Compound 8, containing a benzyloxime ether moiety in its sidechain, was the only one that exerted a protective effect at a higher, 10 μM concentration, while at lower (10 nM– 1 μM) concentrations it promoted tBHP-induced cellular damage. Brain endothelial cells were protected from tBHP-induced barrier integrity decrease by treatment with 10 μM of compound 8, which also mitigated the intracellular reactive oxygen species production elevated by tBHP. Based on our results, 17-oxime ethers of oxidized ecdysteroids modulate oxidative stress of the BBB in a way that may point towards unexpected toxicity. Further studies are needed to evaluate any possible risk connected to dietary ecdysteroid consumption and CNS pathologies in which BBB damage plays an important role.


Introduction
Ecdysteroids are insect molting hormone analogs widespread in the Plant Kingdom, and they have attracted a significant interest due to their non-hormonal anabolic, cytoprotective, and vascular protective activity in mammals.Several recent clinical trials have set their focus on these compounds as potential therapeutic agents in the treatment of sarcopenia (NCT03021798, NCT03452488), or the frequently fatal respiratory deterioration in COVID-19 (NCT04472728).20-Hydroxyecdysone (20E), the most abundant representative of this compound group, was previously found to exert neuroprotective activity in rodent models of cerebral ischemia/ reperfusion [1,2].Further, we have recently reported a set of three new, highly oxidized ecdysteroids to protect human brain endothelial cells from oxidative injury [3].
The chemical modification of lipids, proteins, and DNA by reactive oxygen species (ROS) can result in cellular and tissue damage, implicating oxidative stress in the pathogenesis of numerous diseases and injuries affecting almost every organ system.Although oxidative stress and its related disorders are more prevalent in older individuals, environmental factors such as air pollution and UV exposure can expedite the development of these conditions in people of all ages [4].The impact of oxidative stress on neurodegenerative diseases is of significant interest, as it has been linked to the severity of disease pathology.Biomarkers such as peroxiredoxins and ubiquinone/ubiquinol are found to be elevated in individuals with Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, and are associated with cognitive impairment [4][5][6].The blood-brain barrier (BBB) plays a crucial role in ROS-mediated injury and neurodegenerative diseases.This barrier is composed of endothelial cells that have a strong and dynamic interaction with the neighbouring cells pericytes and astrocytes.The brain endothelial cells have a unique protection system and it controls the transport of substances in and out of the brain via tight junctions, transport pathways, and efflux proteins [7].Given that ROS can affect brain endothelial cells and cause BBB disruption, it is crucial to explore whether compounds such as, e.g., ecdysteroids, can provide protection to these cells, promoting BBB protection in the early stages of neurological diseases.
The cytoprotective effect of 20E is at least partly due to its ability to activate protein kinase B (Akt) [8].In our previous studies on various ecdysteroids as activators of this kinase, we found that calonysterone (1), and particularly its side-chain cleaved derivative 2, are more potent in this regard than 20E in muscle skeletal myotubes [9,10].These findings, and the relative ease of performing chemical modifications on the 20-keto group of compound 2 led us to select it as a starting point to create a new cohort of potentially cytoprotective semi-synthetic ecdysteroid derivatives.
Our previous studies on ecdysteroid oximes and oxime ethers revealed that poststerone, the side-chain cleaved derivative of 20E, can be transformed into 20-oximes and oxime ethers in a regioselective manner [11].This opened way to a synthetic strategy to prepare ecdysteroid derivatives with a modified, nitrogen-containing side-chain.
Inspired by the neuro-and cerebrovascular protective activity of natural ecdysteroids against ROS, in this work it was our aim to prepare a set of sidechain-cleaved and oxime ethercontaining sidechain-modified derivatives of calonysterone (1), and to evaluate the compounds' bioactivity as potential BBB protecting candidates.

Chemistry
Oxidative sidechain cleavage.The regioselective oxidative cleavage between the 20,22-diol to eliminate the sterol side chain at the C-17 position of calonysterone 1 was carried out with the hypervalent iodine reagent (diacetoxyiodo)benzene (PIDA), which had been successfully used for the similar purpose in the case of 20-hydroxyecdysone [10].According to our previous results, using this reagent leads to a significantly better yield than [bis(trifluoroacetoxy)iodo]benzene (PIFA), a more aggressive oxidant [10].Full conversion was achieved within an hour.After neutralization and evaporation of the solvent, normal-phase chromatography was used for purification; this was a more practical choice than reverse-phase separation due to its higher loading capacity and milder solvent evaporation conditions.Outline of the reaction is shown in (Fig 1).
Regioselective formation of 20-oxime or -oxime ether function.Previously, we reported that the 6-enone function of poststerone is relatively less reactive for oxime formation than its 17-oxo group [12], therefore it was postulated that a similar regioselective oximation should be straightforward also for compound 2.
At first, we performed small-scale (with approx.10 mg of substrate) test reactions monitored by TLC in every 10 minutes, and we found that all reactions reached full conversion within 40 minutes.Our experiments included the use of either pyridine or ethanol as solvent, and our experience showed that the reactions proceeded in both solvents with nearly identical results.Therefore, we chose ethanol considering its lower boiling point that makes it easier to evaporate during the work-up.After the transformations, the solvent was evaporated on a rotary vacuum evaporator and liquid-liquid extraction was performed with water and ethyl acetate.Outline of the synthesis and structure of the products is shown in (Fig 2).
Following this strategy, a total of six new ecdysteroid C-20 oxime and oxime ether derivatives were synthesized from larger-scale aliquots of compound 2.After pre-purification of the synthesized materials, their HPLC chromatograms were recorded, which was accompanied by the mapping of the eluent systems for their preparative RP-HPLC purification.To improve sample solubility for preparative RP-HPLC, a 3:7 (v/v) ratio solvent mixture of dimethyl sulfoxide (DMSO) and acetonitrile was used.
Structure elucidation.We have recently reported the structure elucidation and complete  obtained HRMS data verified that our synthetic oximation procedure was regioselective in each case, and the reaction took place at either the 6-or 20-carbonyl groups of the substrate.The location and identity of the newly formed functions was determined by means of comprehensive one-and two-dimensional NMR methods using widely accepted strategies [13,14].
1 H NMR, 13 C DeptQ, edHSQC, HMBC, one-dimensional selective ROESY (Rotating frame Overhauser Enchancement Spectroscopy) spectra (τ mix : 300ms) were utilized to achieve complete 1 H and 13 C signal assignment.It is worth mentioning that due to the molecular mass of compounds 3-8 (374-464 Da) the signal/noise value of the selective ROE experiments strongly exceeds that of the selective NOEs. 1 H assignments were accomplished using general knowledge of chemical shift dispersion with the aid of the 1 H-1 H coupling pattern. 1 H and 13 C chemical shifts (600 and 150 MHz, respectively), multiplicities and coupling constants of compounds 3-8 are compiled in (Table 1).Since the stereostructure of the steroid frame is identical within these compounds, we described the multiplicity and J coupling constants only for 3.The characteristic NMR (S1-S20 Figs in S1 File) and HRMS (S21-S26 Figs in S1 File) spectra of compounds 3-8 are presented as Supporting Information.To facilitate the understanding of the 1 H and 13 C signal assignments, the compounds' structures are also depicted on the spectra.
Only one set of signals appeared in the 1 H and 13 C NMR spectra of each compound, indicating that the regioselective oximation led to the isolation of one stereoisomer for each.The measured Δδ 55 ppm diamagnetic change of δC-20 (211 !156 ppm) supported the C═O !C═NOR conversion [11].During the NMR study of isomeric Z/E 6-oxime derivatives of 20-hydroxyecdysone 2,3;20,22-diacetonide the chemical shift of α carbon atoms (δC-5 and δC-7) in the syn position with respect to the oxime hydroxyl group exhibits a significant (Δδ syn-anti ~5 ppm) diamagnetic shift, which was successfully utilized for differentiation of (Z/ E) isomers [12].In the present case for compounds 3-8, due to the absence of exact data of Δδ syn-anti parameters for the C-21 and C-17 signals, the unambiguous identification of the E/Z isomerism in this way was not possible.To overcome this problem, we utilized a series of selective ROESY experiments on the CH

Biology
Impedance measurements.As a simplified model of the BBB the hCMEC/D3 human brain endothelial cell line was used.This widely employed and well characterized model [15,16] expresses many important BBB characteristics including junctional proteins, metabolic enzymes, efflux and influx transporters [17].We evaluated the effect of the compounds on the viability of hCMEC/D3 cells using impedance measurements.Initially, we tested concentration ranges of 0.01-10 μM for all compounds, and no notable changes in cell viability were observed, except for compounds 3, 4 and 8 (Supporting Information, S27 Fig in S1 File).Although we monitored all concentrations for 24 hours, we observed that the onset of the effects for all compounds was at the 4-hour time point.Therefore, we have focused our results on the 4h time point.For compound 3 we could observe a significant cell index decrease for 10 μM concentration, however, for compound 4 a significant increase for 1 μM concentration was observed (Supporting Information, S27 Fig in S1 File).Notably, compound 8 exhibited the highest and most significant activity.At concentrations of 0.01, 0.1, 1, and 10 μM, it demonstrated a positive effect on barrier integrity.As compound 8 demonstrated the highest activity, we decided to investigate whether it also promotes a protective effect against oxidative stress.Excessive ROS resulting from oxidative stress can cause disruption of the BBB by compromising the antioxidant defense system.The damaging effects of ROS on cellular components such as proteins, lipids, and DNA can lead to the modulation of tight junctions, activation of matrix metalloproteinases, and upregulation of inflammatory molecules, all of which can contribute to BBB damage [18].Tert-butyl hydroperoxide (tBHP) is known to induce cellular damage by generating high levels of ROS [19].Therefore, to assess the protective effects of the compound against ROS-induced damage, we treated cells with tBHP (350 μM) alone or in combination with 0.01, 0.1, 1 and 10 μM of compound 8.We identified the optimal concentration of tBHP by testing different concentrations and selected 350 μM, which did not decrease the cell index below ~50% in our previous work.This concentration was then used for the cell viability assay.We can observe a significant decrease in cell viability by a total of ca.60% in the presence of tBHP compared to the control group (Fig 3B and 3C), indicating tBHP-induced oxidative damage on the cells.Treatment of 10 μM of compound 8 resulted in a significant and steady increase of cell impedance, i.e., it was able to protect the cells efficiently from the harmful effects of tBHP.These findings suggest that the compound might have a protective effect against cellular damage induced by ROS (Fig 3B and 3C).However, at smaller, 10 nM, 100 nM, and 1 μM concentrations a surprising opposite effect was observed.At lower concentrations for 6h or longer incubation, compound 8 increased tBHP-induced toxicity, leading to a disruption of the cellular layer (Fig 3C).We also tested compounds 4 and 6 (3 and 10 μM) in combination with 350 μM of tBHP, and both significantly increased oxidative damage at these concentrations (Supporting Information, S28 Fig in S1 File).
The use of impedance-based monitoring to assess brain endothelial cell function is crucial as it not only measures the number of viable cells but also provides valuable information on the integrity of the cell layer and the extent of barrier damage.This method has been shown to be relevant to evaluate barrier integrity and the overall health of brain endothelial cells [3,20,21].There is evidence indicating that oxidative stress plays a crucial role in the induction of BBB damage [7].The present study provided evidence that treatment with tBHP resulted in brain endothelial damage, which was manifested by a decrease in cell and barrier integrity in certain concentrations of the compounds tested.However, co-treatment with compound 8 significantly altered this effect, leading to the prevention or promotion of oxidative barrier damage.During the 24h-long monitoring of the cell index, a clear concentration-dependent distinction could be made between the protective or damaging effect.Since no data are available on the pharmacokinetics of compound 8, it is not possible to evaluate if a 10 μM plasma level is a realistic concentration in vivo or not.On the other hand, the low-concentration effect of compound 8 to sensitize the BBB to oxidative stress clearly raises a warning concerning its value as a lead compound.
In the broader context, it may be worth stressing that the herein reported compounds are semi-synthetic ecdysteroids that contain oxime ether moieties in their sidechain.This functional group is not expectable to occur in natural ecdysteroids or their metabolites, therefore our results do not directly imply any risk connected to phytoecdysteroid consumption.In our previous study on minor phytoecdysteroids [3], only protective effects were observed.Nevertheless, considering that hardly anything is known about ecdysteroids' bioactivity in relation with the BBB, further studies are needed to evaluate related drug discovery potential and/or risks of this compound family.
Reactive oxygen species measurement.To complete the data, ROS production was also measured in human brain endothelial cells after 0.01-10 μM compound 8 treatment alone or in combination with tBHP for 4 hours.It was confirmed that 350 μM tBHP significantly increased the ROS production, four times to the level of the control group (Fig 4).Addition of 10 μM of compound 8 significantly decreased the tBHP-induced ROS production compared to tBHP treatment alone, although it was still 3 times higher than the control.Treatment with compound 8 alone did not affect ROS production.Compound 8 at lower concentrations (0.01-1 μM) did not decrease ROS production except for 0.1 μM treatment which had a small but significant protective effect against the tBHP induced stress.Barrier integrity tests.To confirm the protective effect of compound 8 at 10 μM concentration against tBHP-induced ROS damage transendothelial electrical resistance (TEER) and permeability measurements for fluorescent marker molecules were performed.For the permeability assay paracellular permeability marker 4 kDa FITC-dextran (FD4) and Evans bluelabeled albumin (EBA, 67 kDa), a marker of adsorption-mediated transcytosis, were used.Changes in intercellular connections were visualized by immunocytochemistry for cell junction associated molecules zonula occludens-1 (ZO-1) and β-catenin (Fig 5).All experiments were performed at the 4-hour treatment endpoint.We found that tBHP decreased the TEER and increased the FD4 permeability of the brain endothelial cell monolayer.This barrier integrity decreasing effect was rescued by the co-incubation with 10 μM compound 8. Treatment with tBHP alone or in combination with compound 8 had no significant effect on the EBA permeability.Compound 8 alone decreased the permeability of the BBB model both for FD4 and EBA compared to the control indicating a barrier tightening effect.These data corroborate the impedance measurements performed with compound 8 alone (S27 Fig in S1 File).After the barrier integrity measurements cells were fixed and stained for junctional proteins.Staining for ZO-1 and β-catenin in the control group showed elongated cell shape and a confluent cell layer typical for hCMEC/D3 cultures.The morphology of brain endothelial cells treated with compound 8 was similar to the control.The barrier integrity decreasing effect of tBHP was also visible on the cellular morphology: irregular cell borders and discontinuity in the staining could be observed.Co-treatment with compound 8 resulted in the recovery of the brain endothelial cell morphology (Fig 5).

Materials and methods
Chemistry.All solvents and reagents were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and were used without any further purification.The progress of the reactions was monitored by thin layer chromatography (TLC) on Kieselgel 60F254 silica plates purchased from Merck (Merck KGaA, Darmstadt, Germany).The examination of the plates was carried out under UV illumination at 254 and 366 nm.
The purification of calonysterone was performed by centrifugal partition chromatography on a 250 ml Armen Spot instrument (Gilson Inc., Middleton, WI, USA).The flash chromatographic purification of compound 2 was carried out on a Combiflash Rf+ instrument (Teledyne ISCO, Lincoln, NE, USA) equipped with diode array and evaporative light scattering detection (DAD-ELSD), and commercially available prefilled RediSep columns (Teledyne ISCO, Lincoln, NE, USA) were utilized.For the analysis of the compounds, we used a dualpump Jasco HPLC instrument (Jasco International Co. Ltd., Hachioji, Tokyo, Japan) equipped with an "MD-2010 Plus" PDA detector.The analytical-scale separations were performed on a Phenomenex Kinetex Biphenyl 100A 5μ 250x4.6 mm (Torrence, CA, USA) HPLC column.The separation of compound 4-8 was performed on an Armen "Spot Prep II" preparative chromatographic apparatus (Gilson Inc., Middleton, WI, USA) equipped with a dual-wavelength UV detector and four individual solvent pumps.The RP-HPLC purification of the ecdysteroid products was carried out with adequately chosen isocratic eluent mixtures of acetonitrile and water.

Isolation of calonysterone (1).
A commercially available extract prepared from Cyanotis arachnoidea roots was purchased from Xi'an Olin Biological Technology Co., Ltd.(Xi'an, China) [22], and subjected to a chromatographic purification to obtain the starting material calonysterone (1) as published before [23].Briefly, 5.46 kg of extract was percolated with 15.5 L of methanol, and after evaporation of the solvent, the dry residue (700 g) was subjected to further separation by a multi-step chromatographic fractionation through silica gel.The final purification of 1 was carried out using centrifugal partition chromatography in ascending mode with a biphasic solvent system of n-hexane-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v) [23].
Preparation of compound 2 via the oxidative side-chain cleavage of calonysterone (1).An aliquot of 2 g of calonysterone (1) was dissolved in 500 ml of methanol.One equivalent (1.34 g) of PIDA was added, and the reaction mixture was stirred for 60 minutes at room temperature.The solution was then neutralized with 10% aq.NaHCO 3 , and the solvent was evaporated under reduced pressure on a rotary evaporator.Subsequently, the residue was redissolved in acetone and adsorbed on 10 g of silica gel for dry loading.The product was purified by flash chromatography on a 24 g silica column (flow rate 35 ml/min, run time: 60 min) with a gradient of dichloromethane (A) and methanol (B), from 0% to 15% of solvent B in A. The separation afforded compound 2 in a yield of 45.5%.
General procedure for the synthesis of sidechain cleaved calonysterone 20-oxime and oxime ether derivatives 3-8.A 120 mg aliquot of compound 2 (0,34 mmol) was dissolved in ethanol (20 ml) and depending on the functional group to be coupled, 120 mg of hydroxylamine hydrochloride (compound 3) or alkoxyamine hydrochloride (compounds 4-8) was added to the solution under stirring.After 40 minutes of stirring at 80˚C the solution was evaporated to dryness under reduced pressure.After water addition to the dry residue (100 ml), the aqueous solution was extracted three times with ethyl acetate (3x100 ml) and the combined organic phase was dried over anhydrous Na 2 SO 4 .Subsequently, the solution was filtered, and the solvent was evaporated under reduced pressure.The purification of the mixture was implemented by preparative RP-HPLC to afford the corresponding ecdysteroid product.
Procedures for structure elucidation of the obtained products.HR-MS analysis of the compounds was carried out on an Agilent 1100 LC-MS instrument (Agilent Technologies, Santa Clara, CA, USA) coupled with Thermo Q-Exactive Plus orbitrap spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) used in positive ionization mode.Regarding the samples, 100 μg/ml solutions were prepared with acetonitrile solvent containing 0.1% formic acid. 1 H NMR, 13 C DeptQ, edHSQC, HMBC, and one-dimensional selective ROESY spectra (τ mix : 300ms) were recorded at 295 K on a Bruker Avance III HD 600 (Billerica, MA, USA; 600 and 150 MHz for 1 H and 13 C NMR spectra, respectively) spectrometer equipped with a Prodigy cryo-probehead.The pulse programs were taken from the Bruker software library (Top-Spin 3.5). 1 H assignments were accomplished using general knowledge of chemical shift dispersion with the aid of the 1 H-1 H coupling pattern ( 1 H NMR spectra).DMSO-d 6 were used as the solvent and tetramethylsilane (TMS) as the internal standard and amounts of approximately 1-5 mg of compound was dissolved in 0.1 ml of solvent and transferred to 2.5 mm Bruker MATCH NMR sample tube (Bruker).Chemical shifts (δ) and coupling constants (J) are given in ppm and in Hz, respectively.To facilitate the understanding of the 1 H and 13 C signal assignments, the structures are also depicted on the spectra (Supporting Information, S1-S20 Figs in S1 File).
Impedance measurements for cell viability assays.The impedance of brain endothelial cells was assessed using the real-time cell electronic sensing analysis (RTCA), which has been shown to correlate with cell number, adherence, growth, and viability [25].The hCMEC/D3 cells were seeded in 96-well E-plates (Agilent, USA) with golden electrodes at the bottom of the plate at a density of 5×10 3 cells per well and incubated in a CO 2 incubator at 37˚C for 5-6 days, until cell growth reaches a plateau phase.The medium was changed every two days.Once the cells reached a stable growth, they were treated with compounds 2-8 at concentrations ranging from 0.01 to 10μM, and their impedance was monitored for 24 hours.From the impedance changes we can interpret the effects of the compounds on cell viability, cell adhesion to the plate or the strength of intercellular junctions [26].Triton X-100 was used to determine 100% toxicity.After 24-hours measurement, we found that the compounds exhibited the highest level of activity after 4-hours of treatment.As a result, we decided to focus our treatments at this time point.
Preparation of stock and working solutions for the cellular assays.The compounds were obtained as dry powder and stored at -20˚C until use.Stock solutions were prepared by diluting the compounds in DMSO to a final concentration of 10 mM and stored at -20˚C.Working solutions were freshly prepared by diluting the stock solutions in cell culture medium to obtain a concentration range of 0.01-10 μM.
Induction of oxidative stress by tert-butyl hydroperoxide.The oxidative compound tert-butyl hydroperoxide (tBHP) can cause cell death through apoptosis or necrosis by generating tert-butoxyl radicals via iron-dependent reactions.This results in lipid peroxidation, depletion of intracellular glutathione, and modification of protein thiols, leading to loss of cell viability [19,27,28].To determine a concentration that would result in approximately 50% cell viability loss, various concentrations of tBHP were tested ranging from 1-1000 μM in preliminary experiments [3].Based on these results, 350 μM tBHP was found to be effective and was used in combination with the selected concentrations of the compounds to test for potential protective effects.
Barrier integrity assays.TEER measurements reflect the permeability of intercellular tight junctions for ions [26].Resistance was measured using the EVOM (Endothelial Volt-Ohm Meter) instrument combined with chopstick electrodes (World Precision Instruments, USA).TEER was expressed relative to the surface area (Ω × cm 2 ).TEER values of cell-free inserts (10.41 ±0.48 Ω × cm 2 , n = 16) were subtracted from the measured data and normalized to the control group.Permeability experiments were performed as described previously [29].To measure the flux of paracellular permeability the fluorescent marker FD4 was used.Transcellular permeability was estimated by the transfer of EBA dye across endothelial cell layers.hCMEC/D3 cells were passaged onto cellQuart culture inserts and grown for 5 days.Cells were treated for 4 hours then permeability assays were performed [29].After the assay the fluorescent intensity of the marker molecules in the upper and lower cell culture insert compartments were determined by a microplate reader (Fluostar Optima, BMG Labtech, Germany; emission wavelength: 485 nm, excitation wavelength: 520 nm).Flux across cell-free inserts was also measured.Endothelial permeability coefficient was calculated as described in an earlier publication of the team [30].
ROS measurement.Total ROS generated after the treatments with compound 8 and tBHP alone or in combination was measured by a fluorometric detection probe, chloromethyl-dichloro-dihydro-fluorescein diacetate (DCFDA) assay (Life Technologies, USA) as described earlier [29].DCFDA diffuses into the cells and becomes deacetylated by intracellular esterases yielding a fluorescent molecule, which can be detected (Fluostar Optima, ex.485 nm/ em.520 nm).hCMEC/D3 cells were cultured in black-wall 96-well plates (Corning) until confluency and were treated for 4 hours.In the last 1 hour of the treatment DCFDA was added to the cells and fluorescence was measured in real time.The fluorescent values detected are presented as arbitrary units.
Statistics.The mean ± SD values were used to present the data.The statistical significance between different treatment groups was assessed using one-way ANOVA, followed by Bonferroni's multiple comparison post-tests (GraphPad Prism 9.0; GraphPad Software, USA).At least four parallel samples were used, and changes were considered statistically significant when p < 0.05.

Conclusions
In this study, we have prepared a sidechain cleaved, oxidized ecdysteroid and six of its oxime or oxime ether derivatives.Using a relevant in vitro cellular model for blood-brain barrier integrity, we demonstrated that the compounds have a significant impact on the oxidative stress-resistance of the BBB.At low doses, compound 8 increased t-BHP-induced cellular damage while at a higher concentration it acted as a protective agent.Our results raise a warning that semi-synthetic modifications of cytoprotective ecdysteroids may unexpectedly alter their bioactivity profile towards harmful effects on cerebrovascular endothelial cells, which may confer them a central nervous system toxicity.The significance of these findings concerning phytoecdysteroid consumption is yet unclear and requires further studies.

1 H and 13 C
signal assignment of compound 1[9] and the sidechain-cleaved calonysterone derivative 2[10].Structure elucidation of the new compounds 3-8 (Fig2) was performed based on the molecular formulas obtained by HRMS and on detailed NMR studies.The

Fig 3 .
Fig 3.The effects of compound 8 at concentrations of 0.01, 0.1, 1, and 10 μM treatment on human brain microvascular endothelial cells (hCMEC/D3) were evaluated using impedance-based assays to assess cell viability and barrier integrity in the absence and presence of oxidative stress promoted by tert-butyl hydroperoxide (tBHP).A: Time-dependent impact of 8 on cell viability following co-treatment with tBHP (350 μM).B: Impact of 8 on cell viability at 4 hours co-treatment with tBHP (350 μM).C: Impact of 8 on cell viability at 24 hours co-treatment with tBHP (350 μM).The data are presented as the mean ± standard deviation (SD) and were obtained from a minimum of two independent experiments (n = 2−3) with 3−9 technical replicates.Data analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test.The results were statistically significant with *p < 0.05, ****p < 0.0001, compared to the control group, and #### p < 0.0001, compared to the tBHP group.https://doi.org/10.1371/journal.pone.0290526.g003